Minimally invasive transplantation of iPSC-derived .... Mol. G… · vanilloid receptor TRPV1. We...

Minimally invasive transplantation of iPSC-derivedALDHhiSSCloVLA41 neural stem cells effectivelyimproves the phenotype of an amyotrophic lateralsclerosis model

Monica Nizzardo{, Chiara Simone{, Federica Rizzo, Margherita Ruggieri, Sabrina Salani, Giulietta

Riboldi, Irene Faravelli, Chiara Zanetta, Nereo Bresolin, Giacomo P. Comi{ and Stefania Corti{,∗

Dino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation, University of Milan,

Neurology Unit, IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, Milan 20122, Italy

Received July 15, 2013; Revised and Accepted August 28, 2013

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motorneurons. Currently, there is no effective therapy for ALS. Stem cell transplantation is a potential therapeuticstrategy for ALS, and the reprogrammingof adult somatic cells into induced pluripotent stemcells (iPSCs) repre-sents a novel cell source. In this study, we isolated a specific neural stem cell (NSC) population from humaniPSCs based on high aldehyde dehydrogenase activity, low side scatter and integrin VLA4 positivity.Weassessed the therapeutic effects of these NSCs on thephenotype of ALS mice after intrathecal or intravenousinjections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injec-tion. Compared with control ALS, treated ALS mice exhibited improved neuromuscular function and motor unitpathology and significantly increased life span, in particular with the systemic administration of NSCs (15%).These positive effects are linked to multiple mechanisms, including production of neurotrophic factors and re-duction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of thevanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert atherapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treatingALS and other neurodegenerative diseases.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a neurodegenerativedisease that is characterized by clinical and pathologicalsigns of upper and lower motor neuron degeneration, progres-sive paralysis and precocious death (1). Currently, there are noeffective treatments. The majority of cases are sporadic, and�10% are familial (1). Of the familial cases, �20% arelinked to mutations in the superoxide dismutase 1 (SOD1)gene (2). Although several other causative genes have beenidentified in recent years, with C9ORF72 and TARDBP

among the most common, almost 50% of familial ALS caseshave unidentified genetic aetiology, although this proportionmay vary according to different genetic backgrounds (1). Theaethiopathogenesis of sporadic ALS is largely unknown (3),which hinders the development of clinically meaningful treat-ment (1,4,5).

Expression of mutant SOD1 in rodents causes progressive de-generation of motor neurons, leading to progressive paralysisthat resembles the manifestations of human ALS (6,7). Althoughthe molecular mechanisms leading to progressive neuronaldegeneration are likely multi-factorial, increasing evidence

†These authors contributed equally to this work.‡These authors contributed equally to this work.

∗To whom correspondence should be addressed at: Neuroscience Section, Department of Pathophysiology and Transplantation, University of Milan,Neurology Unit, IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, Via Francesco Sforza 35, 20122 Milan Italy. Tel: +39 0255033817;Fax: +39 0250320430; Email: [email protected]

# The Author 2013. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.For commercial re-use, please contact [email protected]

Human Molecular Genetics, 2014, Vol. 23, No. 2 342–354doi:10.1093/hmg/ddt425Advance Access published on September 4, 2013

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implies a role for non-autonomous cell death as a key event in thedisease pathology (3). In particular, environmental non-neuronal cells such as astrocytes and microglia are involved inthe death of motor neurons (8).

Cell transplantation therapy could be a promising therapeuticstrategy for a number of disease processes. There is accumulat-ing experimental evidence of non-cell-autonomous contribu-tions of cells other than motor neurons to ALS pathology. Inaddition, the disease is characterized by a complex interplay ofseveral pathogenic events, such as protein aggregation, glutam-ate toxicity, RNA dysfunction and inflammatory events. Stemcell transplantation can be therapeutic via multiple mechanisms,including replacement of micro-environmental cells such asastrocytes and neuronal precursors. Furthermore, stem celltherapy can reduce inflammation and neuropathological hall-marks such as neuronal tangles/aggregates, protect motorneurons and neuronal circuitry and ultimately replace degener-ated cells (9).

Our group and others have demonstrated these positive effectsof stem cell transplantation (neural and non-neural cells) (9–15).In 2009, the US Food and Drug Administration approved the firstphase I safety trial of direct intraspinal transplantation of neuralstem cells (NSCs) into patients with ALS; the study is still in pro-gress (9). Recently, transplanted NSCs were demonstrated to ef-fectively slow the disease and prolong survival in ALS mice, andin some cases (25% of treated mice), the phenotype was com-pletely rescued (survival increased to over a full year versus4 months in untreated animals) (10). These data represent an un-precedented therapeutic success in motor neuron disease modelscompared with other pharmacological/molecular treatments.However, 40% of mice injected with NSCs show a very mild in-crease in survival. The reason for this variability is unknown andmay be related to the type of cell transplanted and our incompleteunderstanding of the key variables important for therapeuticsuccess. Human NSCs are derived from primary centralnervous system (CNS) tissue (fetal brain), which is a limitedsource of cells. Furthermore, multiple direct injections of stemcells into the spinal cord will require major invasive surgery ifthis strategy is translated to humans.

The method of cell delivery represents a major hurdle in devel-oping a cell-mediated approach. The cells must be distributedalong the CNS, targeting both lower and upper motor neurons,using a non-invasive method that leads to sufficient engraftment.The method presented here progresses beyond the current limitsof cell therapy, which is based on direct injection into the spinalcord. We utilize a cell population that can efficiently migrate intothe CNS from the cerebrospinal fluid (CSF) or from the bloodafter systemic injection. We previously described that the selec-tion of a specific subset of NSCs can provide a major advantagein therapeutic efficacy with respect to mixed NSC/neuronal pre-cursor populations (11–13). We previously demonstrated posi-tive therapeutic effects of a subset of self-renewing, multipotentrodent NSCs selected for their aldehyde dehydrogenase (ALDH)activity. When intrathecally transplanted into the SMA andSMARD1 mouse models of motor neuron disease, the cellsmigrate into the spinal cord and ameliorate the diseasephenotype (11,13).

In this work, we sought to test the therapeutic potential ofhuman NSCs that were selected for their high ALDH activity,low orthogonal light scattering (ALDHhiSSClo) and expression

of integrin VLA4. The presence of VLA4 in NSCs allows them tocross the blood-brain barrier (BBB), particularly in the presenceof inflammation or a weakened BBB, as in ALS animal modelsand human patients (16,17).

In addition, we wanted to explore minimally invasive admin-istration protocols, such as intrathecal and systemic intravenoustransplantation, in an ALS mouse model. This strategy allowedus to repeatedly deliver the cells, achieving a widespread distri-bution of a significant amount of cells in the CNS. We hypothe-sized that a progressive escalation in cell dosage might lead to aprogressively enhanced therapeutic effect. Thus, we initiated aregimen in which cell dosage was amplified by gradually in-creasing the number of injections over time. We derived theNSCs from non-virally generated induced pluripotent stemcells (IPSCs) that were reprogrammed from skin fibroblasts ofhealthy subjects. This cell source is attractive due to availability,similar pluripotency as embryonic stem cells (ESCs) and noassociated ethical controversy. With this strategy, we achieveda significant improvement in the disease phenotype, evenwhen treating early-mid symptomatic animals.

RESULTS

Isolation and characterization of an ALDHhiSSCloVLA41NSC population from human iPSCs

We used an iPSC line generated in our lab with a non-viral, non-integrating method and fully characterized its pluripotent fea-tures (Supplementary Material, Fig. S1). We differentiatediPSCs into NSCs with a multistage differentiation proceduredescribed previously for human ESCs and iPSCs (15,18)(Fig. 1A). This protocol generates .90% PAX6-positive neuro-epithelial cells. Then, we used FACS analysis to isolate a specificcell fraction based on ALDHhiSSClo properties, as previouslydescribed for murine NSCs (Fig. 1B) (11,13). This fractionwas further selected for the expression of VLA4+ (Fig. 1Band C). ALDHhiSSCloVLA4+ cells are self-renewing andmultipotent, and they also express neural precursor markerssuch as SOX1, SOX2 and nestin (Fig. 1D). Moreover, thesecells can differentiate into the three major neuroectodermallineages (Fig. 1D–G); they differentiate into motor neuronswhen grown in the presence of retinoic acid (RA) and sonichedgehog (SHH) (15) (Supplementary Material, Fig. S2).

Both intrathecally and systemically transplantedALDHhiSSCloVLA41 NSCs engraft in thespinal cord of SOD1G93A mice

To assess the therapeutic potential of ALDHhiSSCloVLA4+NSCs, we evaluated whether the NSCs reached the CNS follow-ing intrathecal injection or systemic administration. For theseexperiments, we used the SOD1G93A mouse model of ALS(Figs 2 and 3). ALDHhiSSCloVLA4+ NSCs (1 × 106 cells)were delivered into the CSF by lumbar puncture in SOD1 miceat 90 days of age, with additional injections at 105 and 120 daysof age (Fig. 2A). For the systemic injection, cells were deliveredbeginning at 90 days of age with weekly injections into the tailvein until the end stage (Fig. 3A). To trace the transplanted cellsin the host spinal cord, we used ALDHhiSSCloVLA4+ cellsexpressing green fluorescent protein (GFP). To assess donor celldistribution and engraftment, the brain and spinal cord were

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collected at 140 days and at the end stage of the disease. Visualiza-tion of ALDHhiSSCloVLA4+ cells in the spinal cord revealedsingle cells in the host parenchyma and cells distributed in clusters(intrathecal model: Fig. 2B; systemic model: Fig. 3B). WhenALDHhiSSCloVLA4+ donor cells were intrathecally or system-ically administered, they engrafted in both grey and white matterregions of the mouse spinal cord. A quantitative analysis of donor-derived cells showed a significantly higher density of donor cellsin the anterior horn, the site of active degeneration. In particular,the anterior horns in the cervical and lumbar regions had highercell densities relative to the thoracic zone (intrathecal model:Fig. 2C; systemic model: Fig. 3C).

Quantitative measurements of donor-derived cells in the spinalcord showed that an average of 6.49+0.55 cells per section in thecervical and 6.7+0.41 cells per section in the lumbar spinal cordwerepresent following intrathecal administration(Fig.2C).A sig-nificant proportion of donor cells maintained an undifferentiated

phenotype, while the rest differentiated into neuronal and gliacells (Fig. 2D and E, Supplementary Material, Fig. S3). ThevastmajorityofcellsexpressGABA,supportiveofa neuronalpre-cursor phenotype or of an interneuronal phenotype. Few cellsdisplay a cholinergic phenotype, supported by their positivityfor ChAT. Glial cells are almost all astrocytes, and only a minorproportion acquired an oligodendrocytic phenotype (,5%) asquantified in Figure 2. As expected for their ectodermal embryon-ic layer commitment, NSCs do not differentiated into microglia.Donor cells also reached the brain and weredetected in the corticaland subcortical brain areas (Supplementary Material, Fig. S4). Noadverse effects, including tumour formation, abnormal cellgrowth or inflammation, were detected in the recipient animals.

In the animals treated with systemic injections, quantificationof donor-derived cells in the spinal cord revealed the presence of8.66+ 0.49 cells per section in the cervical and 8.93+ 0.38cells per section in the lumbar spinal cord (Fig. 3C). We found

Figure 1. Differentiation of iPSCs into NSCs and selection of the ALDHhiSSCloVLA4+ fraction. (A) Scheme of the differentiation protocol of NSCs from iPSCs.Initially, the iPSCs are positive for OCT4. After detaching from the plate, the iPSCs form aggregates that are cultured for 7 days in neural medium. The differentiatedcells show features of neuroepithelial (NE) rosettes. The primitive NE cells were positive for PAX6. These cells were then selected by FACS for their ALDHhiSSCloand VLA4 positivity and expanded in vitro. The cells were cultured with RA and SHH and differentiated into OLIG2-expressing motor neurons. They could be furthermatured into their neuronal phenotype in the presence of neurotrophic factors and reduced concentrations of SHH and RA. (B) Representative flow cytometric analysisof ALDH activity and VLA4+ positivity on iPSC-derived NSCs. NSCs were selected according to forward and side scatter properties. Cells incubated with Aldefluorsubstrate and the specific inhibitor of ALDH (DEAB) were used to establish the baseline fluorescence and to define the ALDHhi region. After sorting, this cell popu-lation is enriched as shown in the right panel. The ALDHhiSSClo fraction was further selected by FACS for VLA4 expression (left panel) and seeded in culture. Scalebar: 70 mm. (C) iPSC cells positive for VLA4 (red; nuclei stained with DAPI, blue signal). (D) Sorted ALDHhiSSCloVLA4+ cells grown at clonal density in NEPmedium form epithelial-like clones positive for NSC markers such as SOX1, SOX2 and nestin. Scale bar: 100 mm. After in vitro differentiation, ALDHhiSSClo cellscould give rise to cells of the three neuroectodermal lineages including GFAP-positive astrocytes (red), O4-positive oligodendrocytes (red) and motor neurons (ChATred, TuJ1, green). Nuclei are counterstained with DAPI (blue signal). Scale bar: GFAP, 100 mm; O4, 50 mm; and TuJ1 and ChAT, 100 mm. (E) Quantification of cellspositive for NSC markers. (F) Quantification of cells showing positive staining for astrocyte and oligodendrocyte markers after appropriate differentiation. (G) Quan-tification of cells that are double-positive for motor neuron markers TuJ1 and ChAT.

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Figure 2. iPSC-derived NSCs migrate and engraft into the spinal cords of SOD1G93A mice after intrathecal and systemic transplantation. (A) Experimental design:GFP-NSCs (1 × 106 cells) were delivered by intrathecal injection into SOD1G93A mice at 90 days of age, with two additional injections at 105 and 120 days.(B) Donor GFP+ cells were detected in the spinal cord, particularly in the anterior horns. (C) Quantification of GFP-donor cells in the cervical, thoracic andlumbar spinal cord. Error bars indicate the SD. (D) Quantification of the phenotype acquired by the donor cells revealed the presence of cells with an undifferentiatedphenotype (nestin), neuronal (NeuN) phenotype and glial cells (GFAP). Error bars indicate the SD. (E) Representative images of cells acquiring a neuronal phenotype,as indicated by positivity for NeuN (red) and GFP (green). Nuclei are counterstained with DAPI (blue signal). Scale bars: (B) 150 mm and (E) 50 mm.


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Figure 3. iPSC-derived NSCs migrate and engraft into the spinal cords of SOD1G93A mice after intravenous transplantation. (A) Experimental design: GFP-NSCcells (1 × 106 cells) were delivered by weekly intravenous injection into SOD1G93A mice starting at 90 days of age. (B and C) Donor GFP+ cells were detected in thespinal cord, particularly in the anterior horns. (C) Quantification of GFP-donor cells in the cervical, thoracic and lumbar spinal cord. Error bars indicate the SD.(D) Quantification of the phenotype acquired by the donor cells revealed the presence of cells with an undifferentiated phenotype (nestin), a neuronal (NeuN) pheno-type and a glial (GFAP) phenotype. Error bars indicate the SD. (E) Representative images of cells acquiring a neuronal phenotype that are positive for NeuN (red) andGFP (green). Scale bars: (B) 150 mm right, 120 mm left; (E) 50 mm upper panel, 75 mm lower panel.


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IgG deposits from blood vessels in the spinal cord of SOD1 micebut not in wild-type mice (Supplementary Material, Fig. S5).These data confirmed the weakness of the BBB in SOD1G93Amice (16,17,19), which allowed transplanted cells to reach thespinal cord after the systemic injection. The phenotype of thecells transplanted by intravenous injection was similar to theintrathecal administration; the engrafted cells maintained an un-differentiated phenotype or differentiated into neuronal and gliacells (Fig. 3D and E, Supplementary Material, Fig. S6). With sys-temic administration, we also detected the presence of donor cellsin the cortical and subcortical brain areas of ALS mice (Supple-mentary Material, Fig. S7). Neuronal undifferentiated cells sur-vived and were detectable until the late stage of the disease,when the animals were sacrificed. No adverse effects wereobserved after systemic transplantation.

Cell transplantation improved disease phenotypeand extended survival of ALS animals

We first evaluated whether both intrathecal and systemic deliv-ery of ALDHhiSSCloVLA4+ cells could ameliorate the pheno-type and extend survival in SOD1G93A mice (Fig. 4). For thephenotypic assessment, mice were tested on the rotarod, andthe survival was recorded.

Intrathecal transplantation of ALDHhiSSCloVLA4+ cellsinto SOD1G93A mice (n ¼ 24) significantly extended survivalby 10 days compared with vehicle-treated animals (n ¼ 25)(vehicle, 143+ 6 days mean survival; ALDHhiSSCloVLA4+,153+ 5 days; x2 ¼ 6.1, P ¼ 0.0135; Fig. 4B). Systemic trans-plantation of ALDHhiSSCloVLA4+ cells into SOD1G93Amice (n ¼ 16) also significantly extended survival by 23 dayscompared with vehicle-treated animals (n ¼ 24) (vehicle,148+ 7 days mean survival; ALDHhiSSCloVLA4+, 171+ 6days;x2 ¼ 11.81, P ¼ 0.0006; Fig. 4D). The survival differencebetween the intrathecal and systemic group was statisticallysignificant (P ¼ 0,0001). The rotarod test was used to assessneuromuscular function in transplanted and untreated animals.At 4 weeks after starting transplantation, the cell-treatedanimals presented a significant improvement in rotarod perform-ance relative to untreated (P , 0.001) and fibroblast-treatedanimals (P , 0.001) (Fig. 4A and C). Rotarod functionaloutcome was similar between the two groups and the differenceis not statistically significant.

To determine whether ALDHhiSSCloVLA4+ transplant-ation protects against the loss of motor neurons, we quantifiedthe number of motor neurons in spinal cord sections andventral spinal nerve roots. At 140 days of age, motor neuronloss was significantly reduced by cell transplantation via bothintrathecal and systemic administration. Mice transplantedwith stem cells had 40% more motor neurons than vehicle-treated SOD1G93A mice (P , 0.001); this increase likelyaccounted for the observed functional improvement (Fig. 4E).Transplanted animals also presented with a preservation ofaxonal density in the L4 ventral root, while control miceshowed a substantial reduction in axons compared with wild-type mice. Quantitative assessment revealed that in both trans-planted SOD1G93A groups, significantly fewer axons werelost with untreated SOD1 mice (treated versus untreated P ,0.001). Axons in adult L4 ventral nerve roots were preserved,showing an increase of 50% with respect to vehicle-treatedanimals (P , 0.001) (Fig. 4F).

We detected donor GFP cells in the muscle located in the prox-imity of the sarcolemma of the muscle fibres in mice transplantedsystemically at 140 days of age. We hypothesized that neuro-muscular junction (NMJ) preservation may have been involvedin the improved neuromuscular function in the systematicallydelivered group (Supplementary Material, Fig. S8).

Changes in growth factor levels and a reductionin micro- and macrogliosis after cell transplantation

We next investigated the molecular mechanisms that may under-lie the observed therapeutic effect. Using an enzyme-linked im-munosorbent assay (ELISA), we determined that iPSC-derivedALDHhiSSCloVLA4+ NSCs produced a spectrum of neuro-trophic and neurite outgrowth-promoting factors (Fig. 5)(14,15). ALDHhiSSCloVLA4+ NSCs secreted significantamounts of glial cell-derived growth factor (GDNF) (713.8+69.75 pg/ml; P , 0.001 versus fibroblasts), brain-derived neuro-trophic factor (BDNF) (469.67+52.76 pg/ml; P , 0.001),neurotrophin-3 (NT3) (158.46+21.68 pg/ml; P , 0.001) andtransforming growth factor alpha (TGF-a) (123.51+11.42;P , 0.001) (Fig. 5A–D).

In our neuropathological analysis, the CNS tissues ofSOD1G93A ALS mice showed the presence of a significant in-flammatory (microglial) reaction that may play an important rolein the disease pathogenesis (1,10). Interestingly, we demon-strated a significant reduction in macrophage/microglia cellpopulations in both lumbar and cervical spinal cord in treatedmice compared with vehicle-treated and untreated SOD1G93Amice. The number of activated microglia in the ventral hornwas 146.8+ 13.4 per section in vehicle-treated SOD1G93Amice versus 65.4+ 7.9 in the intrathecally treated SOD1G93Amice (n ¼ 6 per group/condition; P , 0.001) (Fig. 5F).Similar results were obtained with systemic injection, with54.9+ 9.6 microglia per section in the treated SOD1G93Amice versus 153.4+ 14.9 in vehicle-treated animals (n ¼ 6per group/condition; P , 0.001) (Fig. 5F).

Astrogliosis also plays an important role in ALS pathogen-esis, particularly in non-autonomous motor neuron death(1,10). Staining for the astrocytic marker glial fibrillary acidicprotein (GFAP) suggested diminished astrogliosis in the spinalcord (particularly in the critical cervical/lumbar regions) ofNSC-engrafted SOD1G93A mice (Fig. 5E). The number ofhypertrophic reactive astrocytes in the ventral horn was254.9+ 24.6 per section in vehicle-treated SOD1G93A miceversus 163.9+ 12.4 in intrathecally treated SOD1G93A mice(n ¼ 6 per group; P , 0.001) (Fig. 5G). In the systemic injec-tion group, we observed a mean of 145.6+ 22.3 GFAP-positivecells compared with 243.7+ 21.8 in the vehicle-treated group(n ¼ 6 per group; P , 0.001) (Fig. 5G).

NSCs inhibit ALS astrocyte activation throughTRPV1 receptor activation

We next analysed the possible molecular mechanisms involved inthe reduction of astrocyte activation/proliferation by NSC trans-plantation (Fig. 6). In a series of in vitro experiments, we demon-strated that stimulating mouse astrocytes with factors releasedfrom iPSC-derived NSCs, but not with factors released fromfibroblasts, significantly reduced the viability of SOD1G93Aastrocytes. NSCs reduce astrocytic tumour expansion by


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releasing endovanilloids that activate the vanilloid receptor (tran-sient receptor potential vanilloid subfamily member-1, TRPV1)on astrocytic cells (20). Therefore, we investigated whethersimilar events occurred during ALS. First, we demonstratedthat astrocytes from the CNS of SOD1 mice express TRPV1(Fig. 6A). We found that NSCs are toxic for astrocytes when

grown in co-culture (Fig. 6B). We further showed that blockingTRPV1 with the selective antagonists iodoresiniferatoxin(I-RTX, 10 nM) or capsazepine (CZP, 1 mM) decreased the cyto-toxicity of NSCs on astrocytes (Fig. 6C). The toxic effect of NSCson astrocytes was selectively restricted on the SOD1 background,while non-transgenic and ALDHhiSSCloVLA4+ NSC-derived

Figure 4. Transplantation of ALDHhiSSCloVLA4+ NSCs improves neuromuscular function, increases survival and reduces motor neuron and axon loss in ALSmice. (A and C) Transplantation of NSCs significantly improved motor performance in SOD1 mice, as demonstrated by the rotarod test both in the intrathecally trans-planted group (A) and in systemically injected mice (C) (4 weeks after transplantation, P , 0.001, ANOVA). (B and D) Kaplan–Meier survival curves for mutantSOD1 mice treated intrathecally (B) or systemically (D) with ALDHhiSSCloVLA4+NSCs or with vehicle. Survival was significantly extended for NSC-transplantedmice compared with vehicle-treated mice for both treatment groups (P , 0.05, log-rank test). (E) The motor neuron count (n ¼ 6 for each group) in the lumbar spinalcord of NSC-transplanted, vehicle-treated SOD1 mice and wild-type mice (data represent the mean+SD of the number of motor neurons per section) at 140 days ofage. The evaluation revealed significantly increased numbers of surviving motor neurons in treated SOD1G93A mice (P , 0.001, ANOVA). (F) Quantification ofaxons (data represent the mean+SD) at 140 days of age (n ¼ 6 for each group) demonstrated that transplanted SOD1G93A mice showed a significantly increasednumber of axons (P , 0.001, ANOVA).


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astrocytes were not affected (Fig. 6D). Thus, we conclude thatdonor NSCs reduce reactive astrocytes in the CNS of SOD1mice by activating TRPV1.

DISCUSSION

In this study, we found that repeated intrathecal or systemicinjection of a specific NSC subpopulation ameliorates the ALS

disease phenotype in a mouse model. These results, combinedwith the minimally invasive nature of the delivery method,support the rationale and utility of this approach for patientswith ALS. The beneficial effects of engrafted ALDHhiSSCloVLA4+ NSCs include improved neuromuscular functionand survival, neuroprotection and positive host-environmentmodifications at the neuropathological level.

Stem cell transplantation isgaining increased interestas a thera-peutic strategy because its multifaceted impact can counteract a

Figure 5. NSCs produce neurotrophins and reduce micro- and macrogliosis in SOD1G93A mice. The amounts of (A) GDNF, (B) BDNF, (C) NT3 and (D) TGF-asecreted by the NSCs were determined by ELISA, which demonstrated a significant production of these neuroprotective substances with respect to fibroblasts.(E) Expression of GFAP (red) was reduced in the spinal cord of transplanted SOD1 mice. Nuclei are stained with DAPI (blue). Scale bar: 70 mm. NSC transplantationsignificantly reduced the presence of microglial cells (F) and astrocytes (G) in the spinal cord of intrathecally and systemically injected mice (data represent themean+SD, ∗significant, P , 0.001, ANOVA).


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number of processes that underlie disease pathogenesis. Molecu-lar and pharmacological strategies that focus on a single or fewtargets have been minimally successful in ALS clinical trials,likely because they address only one aspect of this complex neu-rodegenerative disease. In contrast, several experimentalapproaches of NSC transplantation have produced positiveeffects on motor neuron disease in animal models (9–15).

Among the potential cell sources, our population ofALDHhiSSCloVLA4+ stem cells/precursors is an attractivechoice for both reproducibility and the lack of controversyregarding their collection; they can be derived from autologous

or donor fibroblasts, reprogrammed into iPSCs, and grownin vitro. In addition, they can migrate from the CSF or blood tothe spinal cord parenchyma. This property allows non-invasivetransplantation, which is important for clinical translation topatients. Furthermore, these cells present robust survival in thehost CNS and do not trigger side effects such as tumours or ab-normal cell growth. Indeed, we showed that the donor cells ap-propriately migrate and localize throughout the entire spinalcord, particularly in the specific areas that are affected by thedisease: the cervical/lumbar enlargement, the anterior hornsand the areas around endogenous motor neurons. The donorcells were also located in cortical brain areas, thus positivelyinfluencing the upper motor neurons. Of importance, donorALDHhiSSCloVLA4+ cells maintained their stem cell/precursor phenotype or differentiated into neuronal and glialcells, changing the spectrum of cell phenotypes in the host tissuemicroenvironment.

Regarding the mechanisms of cell migration from the CSF andblood toward the parenchyma, the neuronal death that occurs inALS could be a signal that promotes NSC attraction into theCNS. Indeed, the presence of an altered BBB in ALS mice andpatients can promote cell migration (17). VLA4 is a surfaceprotein that mediates transendothelial migration. It is expressedon some haematopoietic cell fractions, in particular on T lym-phocytes, and mediates their ability to cross the BBB (21).A humanized monoclonal antibody against VLA4, natalizumab,is used to treat multiple sclerosis because it blocks the migrationof T lymphocytes into the CNS (21). In contrast, the presence ofVLA4 on NSCs gives them the ability to cross the BBB, particu-larly in the presence of inflammation or weakened BBB, asdemonstrated in humans and animal models of ALS (16,17).

ALDHhiSSCloVLA4+ NSC-treated SOD1 mice presentedan amelioration of the motor neuron phenotype, demonstratedby neuromuscular function tests and increased survival. Trans-plantation of ALDHhiSSCloVLA4+ cells significantly pro-longed survival compared with vehicle-treated animals. Theincreased survival time, although limited, is relevant consideringprevious reports of NSC transplantation in rodent models of ALSthat also showed a transient improvement in motor performanceand a modest increase in survival (22). Recently, the Teng groupreported a remarkable survival increase of 200 days in 40% or upto a full year in 20% of SOD1 animals injected with NSCs at foursites of the spinal cord during the presymptomatic stage ofdisease (10). However, the remaining 40% of mice showed avery mild increase in survival, demonstrating a large variabilityin outcome with unknown cause. The transplantation methodused by Teng et al. (10) required a direct intraparenchymal injec-tion at multiple sites. This procedure is more invasive than theintrathecal and systemic injections that were used in this study.One possibility for the difference in benefit obtained with themore invasive cell therapy by Teng et al. (10) can lie in thenumber of NSCs that successfully engrafted the parenchyma.However, a direct comparison cannot be performed becausethe authors do not present a quantitative analysis of engrafteddonor cell number, even if we can assume indirectly fromother data, such as images of the treated mouse spinal cord,that the number of cells was higher than what we achieved inour experiments. Indeed, both our group and Teng’s groupobserved a positive correlation between the number of engraftedcells and the positive outcome.

Figure 6. NSC-mediated astrocyte suppression by activating endovanilloidreceptor TRPV1. (A) SOD1G93A astrocytes (GFAP, green) are immunopositivefor TRPV1 (red). A single cell is magnified on the left, and pixels withco-localized staining in a single optical section are shown. Scale bars: 40 mmon the left, 10 mm in the higher magnification panel. (B) The viability ofSOD1G93A astrocytes is reduced after stimulation with NSC-conditionedmedium (NSC) but not after stimulation with non-conditioned medium (Ctrl).(C) The NSC-induced cytotoxicity of astrocytes was blocked by the TRPV1 se-lective antagonists I-RTX and CZP. (D) The toxic effect of NSCs on astrocyteswas present on SOD1 astrocytes, while non-transgenic mouse astrocytes andon ALDHhiSSCloVLA4+ NSC-derived astrocytes were not significantlyaffected. All data are mean+SD. ∗P , 0.05, ∗∗P , 0.005 by Student’s t-test.


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The strategy of repeated delivery of the cells allowed achiev-ing a widespread distribution of a significant amount of cells inthe CNS. We hypothesized that also in clinical translation, thetherapeutic effect would be proportional to the number ofengrafted cells that can be achieved with multiple injections.Regarding the risks of raising an immunoresponse with this strat-egy, differentiated cells derived from iPSCs, including NSCs,present a low immunogenic profile (23,24). Moreover, theCNS is a low responding immunological site. In any case, immu-noresponse stimulated by repeated injection of NSCs can be con-trolled by immunosuppressant agents, as we achieved here inmice; no signs of immunorejection were observed. VLA4 is aphysiological antigen expressed in human cells, so it will not ini-tiate an immune response in humans.

We demonstrated an increased therapeutic effect when thecells were administered by systemic injection compared withintrathecal injection. We hypothesize that, with a systemic injec-tion, donor NSCs can exert a positive role both locally and per-ipherally. After entering the CNS via cell adhesion molecules(such as VLA4) and chemokine receptors, systemically injectedNSCs accumulate in perivascular CNS areas and in the areas ofactive degeneration. As a consequence, transplanted NSCssurvive and promote neuroprotection by releasing neurotrophicfactors in situ. There is further evidence suggesting a peripheralimmunomodulatory effect of the NSCs (25). Systemicallyinjected NSCs also enter peripheral organs (e.g. draininglymph nodes and spleen), where they accumulate at the bound-aries of blood vessels. Here, they interact closely with lympho-cytes and professional antigen-presenting cells, impairing theirmaturation and functional activation (25). Indeed, we hypothe-size a peripheral effect of NSCs, which secrete neuroprotectivefactors at the skeletal muscle level, on the maintenance ofNMJ integrity; however, experimental demonstration is stillneeded. Accumulating evidence suggests that clinical ALS ispossibly a multi-organ disease. Although motor neurons are se-lectively affected in ALS, the possibility that ALS might be partof a systemic disease in which motor neurons are especially vul-nerable has been hypothesized. In fact, a number of well-established abnormalities in ALS occur in the immune system,skin, skeletal muscle tissue and lipid metabolism (see forreview26). Moreover, we have to consider that the systemicdelivery relies on multiple injections respect to the intrathecaldelivery over the same period of time and the maintenance ofthe NSCs presence may have been more continuous for the sys-temic route than for the other one, even if at 140 days and at theend stage of the disease the difference in NSC integrationbetween the two routes of delivery was only slightly different.

We hypothesize that the therapeutic effect of ALDHhiSSCloVLA4+ cells in our study relies on multiple mechanismsthat include both neuroprotection and reduction of macro- andmicroglia. NSCs constitutively secrete various neurotrophicand neuroprotective molecules that can exert a synergisticeffect superior to the administration of a single molecule.These factors seem to protect host motor neurons and functionalneuromuscular units, reduce macrophage/microglial infiltrationand decrease endogenous astrogliosis (10,22). In particular, weobserved an increase in the production of GDNF, which canexert a direct neuroprotective effect on endogenous cells. Themodest effects of neurotrophic factor treatment on lifespan inprevious clinical studies of ALS suggest several explanations

including that they are physiologically effective but limited bypharmacokinetic constraints (27).

Indeed, NSCs constitutively secrete more than one neuro-trophic and neuroprotective factor. We also demonstrated a re-duction in the levels of activated macro- and microglia aftercell transplantation, suggesting a potential anti-inflammatoryeffect of ALDHhiSSCloVLA4+ cells. Even if wild-type astro-cytes can exert a positive effect on motor neurons, we andothers (10) have demonstrated that the reduction in astrocytosisplays a part in the therapeutic effect achieved by cell transplant-ation. Therefore, a reduction in astrocytes can be considered ashift of the ALS neuropathological features towards a physio-logical wild-type pattern. Our results suggest a novel mechanismin which NSCs reduce macrogliosis by activating the vanilloidreceptor. Thus, NSCs not only provide cells of the three neuroec-todermal lineages to the host cell environmental, but they canalso influence the behaviour of endogenous cells. Identifyingthe full range of variables underlying the success of cell trans-plantation is essential for confirming the reproducibility of thetherapeutic response for clinical application.

In the present work, we described a feasible and efficientprotocol for the generation of NSC cells from iPSCs that are scal-able for producing highly pure populations of functional iPSC-derived NSCs sufficient for clinical translation. Converting thecell dose used in a mouse to a dose based on surface area (28),we can estimate that for humans we would have to transplantan approximate dose of 1 × 108 cells/m2 that for a man of60 kg (with a body surface area of 1.6 m2) would be 1.6 × 108.Given their indefinite self-renewal, with a doubling timebetween 24 and 48 h, human iPSCs have the potential to yieldan unlimited supply of NSCs.

Our results provide evidence that ALDHhiSSCloVLA4+NSCs derived from iPSCs may represent an effective approachfor the treatment of ALS. We demonstrated that the beneficialeffects observed after stem cell transplantation arise from mul-tiple events that counteract multiple aspects of the disease; thisfeature is crucial for multifaceted disease such as ALS. A com-bination of therapeutic approaches that target different patho-genic mechanisms of the disorder, including pharmacology,molecular therapy and cell transplantation, will increase thechances of a clinically successful therapy for ALS.

MATERIALS AND METHODS

Generation of iPSCs

Human skin fibroblasts were reprogrammed as previouslydescribed (15) using oriP/EBNA1-based episomal vectors en-coding the human genes OCT4, SOX2, NANOG, LIN28,c-Myc and KLF4. After nucleofection, fibroblasts were platedonto 3 × 10 cm dishes covered with Matrigel (BD Biosciences)in fibroblast culture medium, which was changed every otherday. Four days after transfection, the fibroblast culturemedium was replaced with human ESC culture medium(mTeSR, Stemcell Technologies Inc.) for 8–10 days. At day18 after transfection, the first colonies with an iPSC-like morph-ology could be identified. We then picked the iPSC colonies thatwere morphologically similar to ESCs for further analysis andexpansion, as previously described (15).


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Differentiation of iPSCs and selection of theALDHhiSSCloVLA41 fraction

We differentiated iPSCs into neuroepithelial cells using a previ-ously described differentiation protocol (15). iPSCs were platedin neuronal medium composed of Dulbecco’s modified Eagle’smedium/F12 (Gibco, Invitrogen). The medium was supplemen-ted with MEM non-essential amino acids solution, N2 andheparin (2 mg/ml; Sigma-Aldrich) to induce the neuroepithelialcommitment. Cells were then selected by FACS for their ALDH-hiSSClo properties, as previously described (11,13). The iso-lated cells were further selected by FACS for their VLA4positivity (anti-human VLA4, BD) using an appropriate nega-tive control with cells labelled only with the same isotypic Ig.

For cell expansion and clonal culture, ALDHhiSSCloVLA4+cells were plated in a previously described growth medium (NEPmedium) containing FGF with or without EGF (13). Differenti-ation of cultured ALDHhiSSCloVLA4+ cells was induced byplating on poly-lysine/laminin-coated dishes (poly-lysine at20 mg/ml in DPBS; Sigma) and reducing the FGF concentrationwith the addition of RA (1 mM). To induce the motor neuronalphenotype, NSCs were exposed to additional RA (0.1 mM;Sigma-Aldrich) for neural caudalization. After 7 days, we col-lected the posteriorized neuroectodermal cells and suspendedthem for a week in the same medium with RA (0.1 mM) andSHH (100–200 ng/ml; R&D Systems Inc.). On day 24, weadded the growth factors BDNF, GDNF and insulin-likegrowth factor-1 (10 ng/ml each; PeproTech).

Immunocytochemistry of iPSCs and their derivatives

Cells were fixed in 4% paraformaldehyde (PFA) for 10 min, per-meabilized with 0.5% Tween-20 in phosphate-buffered saline(PBS) and exposed to 0.1% Tween-20 with 10% horse serum.We incubated the cells with primary antibodies overnight andwith secondary antibodies for 1 h (Alexa Fluor, Invitrogen).We used the following primary antibodies: SSEA-3 (1:100, Che-micon), SSEA-4 (1:500, Chemicon), TRA1-60 (1:500, Chemi-con), TRA1-81 (1:500, Chemicon), Pax6 (1:200, Millipore),nestin (1:200, Millipore), Sox1 (1:1000, R&D,), SOX2(1:1000, Millipore), TuJ1 (1:200, Millipore), GFAP (1:300,Sigma), Islet1/2 (1:200, Millipore), HB9 (1:200 Millipore),ChAT (1:200, Millipore), MAP2 (mouse monoclonal, 1:100;Sigma), O4 (10–20 mg/ml, Millipore) and Musashi (1:200,Novus Biological). The HB9::GFP gene reporter was used toidentify motor neuron cells, as previously described (29). Forall imaging, we used a confocal LEICA LCS2 microscope.

Animal models

We used transgenic mice of the strain B6.Cg-Tg(SOD1-G93A)1Gur/J, which carries a high copy number of the mutanthuman SOD1 allele containing a Gly93Ala (G93A) substitution.Progeny for experimental analyses was obtained by breedingSOD1G93A transgenics with C57BL/6 wild-type mice. Trans-genic mice were identified by polymerase chain reaction, as pre-viously described (14). All animal experiments were approvedby the University of Milan and Italian Ministry of Healthreview boards and are in compliance with the US National Insti-tutes of Health guidelines (14).

Cell transplantation

Ninety-day-old transgenic SOD1G93A mice were transplantedwith ALDHhiSSCloVLA4+ cells (1 × 106 cells) or vehicleonly (saline) by intrathecal injection (5 ml) (three injections) orby injection through the tail vein (100 ml, weekly injections)(14). These cells were also genetically engineered to expressGFP driven by the cytomegalovirus promoter (15). The cellswere harvested and prepared for transplantation as previouslydescribed (15). The intrathecal group included 24 mice (12males) for transplantation and 25 mice (12 males) in the vehiclecontrol group. The intravenous group included 16 transplantationmice (8 males) and the vehicle group of 24 mice (12 males). Theanimals were evaluated up to the end stage for rotarod perform-ance, survival andhistological evaluationof donorcellphenotype.

The study was designed such that littermates were distributedequally among the transplanted and non-transplanted groups.Three groups of animals (each composed of six mice per timepoint per condition) were euthanized at 140 days of age for histo-logical analysis, quantification of motor neurons and axons andgrowth factor evaluation via ELISA.

Tissue analysis

Animals wereeuthanized,perfused and fixedwith4%PFA inPBS(pH 7.4). The spinal cord was isolated, immersed in PFA for 1 hand then soaked overnight in 20% sucrose in PBS (pH 7.4). Thefixed tissue was frozen in Tissue Tek OCT compound (Sakura)with liquid nitrogen. Tibialis anterior muscles were removedprior flash-frozen in supercooled isopentane. The tissues werecryosectioned and mounted on gelatinized glass slides. Everytenth 20 mm section was collected. All sections were blockedwith 1% fetal calf serum in PBS and permeabilized with 0.25%Triton X-100. Sections were processed for multiple markers to de-termine the cellular phenotype of GFP-labelled cells. Primaryantibodies were added overnight at 48C at dilutions of 1:200 forNeuN (mouse monoclonal antibody, Chemicon), 1:200 fornestin (mouse monoclonal, Chemicon) and 1:200 for GFAP(mouse monoclonal Cy3 conjugate, Sigma) and laminin (rabbitpolyclonal, 1: 500, Sigma-Aldrich).

Mouse and rabbit secondary antibodies conjugated with FITC,RPE, CY3 or biotin (1:200; Jackson ImmunoResearch Laborator-ies, Inc., and Dako) were applied for 1 h at roomtemperature whenunconjugated primary antibodies were used. Anti-GFP antibodyrabbit serum Alexa 488 (1:400 dilution; Molecular Probes) wasused to reveal GFP expression. Co-expression of GFP and tissue-specific markers was evaluated by conventional fluorescence mi-croscopy (Zeiss Axiophot) and laser confocal scanning micros-copy (Leica TCS SP2 AOBS). Optical dissectors and randomsampling were used to obtain an unbiased stereological estimateof the number of GFP-positive cells. To quantify donor cells,we used every tenth coronal section (20 mm) throughout theentire spinal cord (a mean of 25 sections per animal). The numer-ical density of cells was estimated using the optical dissectormethod (14). Optical dissectors sized 100 × 70 × 14 mm wererandomly sampled, and the number of positive cells in each dis-sector was quantified. The density was calculated by dividingthe total number of donor cells by the total volume of optical dis-sectors. The total volume of tissue per specimen (Vcord) contain-ing donor cells was calculated using the Cavalieri method (14).


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This total volume of tissue, multiplied by the number of donorcells per cubic micrometre (Nv), yielded the total number ofdonor cells per specimen (n ¼ Nv × Vcord) (14).

Assessment of motor function and survival

Vehicle- and cell-injected SOD1G93A mice were monitoreddaily following transplantation for clinical signs of diseaseonset. Motor performance was assessed using an acceleratingrotarod device (4–40 rpm Rota-Rod 7650; Ugo Basile,Comerio, Italy). The length of time that the mice remained onthe rotarod was recorded. The investigators performing the be-havioural assessment were blind to the treatment. Mortalitywas scored as the age at which the mouse was unable to rightitself within 30 s when placed on its back, as previouslydescribed (14).

Motor neuron and axon count

Paraffin serial sections (12.5 mm) of lumbar spinal cord wereNissl-stained to quantify motor neuron numbers via light micros-copy, as previously described (14). For the axon count, the tissuewas dissected, immersed in 2.5% glutaraldehyde overnight andpost-fixed in 2% osmium tetroxide. Samples were then dehy-drated in ethanol and embedded in Epon resin. Semi-thin trans-verse sections (1 mm) were stained with toluidine blue. L4 rootswere examined for axon counting on an optic microscope.

Enzyme-linked immunosorbent assay

BDNF, GDNF, NT3 and TGF-a secretion by NSCs was mea-sured by ELISA as previously described (14). The data were ana-lysed using Student’s t-tests.

Analysis of macro- and microgliosis

Histological analysis for quantification of microglia and astro-cytes was performed as described (10).

TRPV1 evaluation and cytotoxicity assay

Detection of the TRPV1 receptor, conditioned medium experi-ments and the cytotoxicity assay were performed as previouslydescribed (20).

Statistical analysis

Kaplan–Meier survival analysis and the log-rank test were usedfor survival comparisons. The rotarod test data were analysedusing an ANOVA followed by a Tukey post hoc analysis for mul-tiple comparisons. The numbers of motor neurons and axonswere statistically evaluated by one-way ANOVA followed bya Tukey post hoc analysis. ELISA results were analysed usingStudent’s t-tests. In culture assays, differences between meanvalues were analysed using a two-tailed Student’s t-test.We used StatsDirect for Windows (version 2.6.4) for all statisticalanalyses, rejecting the null hypothesis at the 0.05 level.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS

We thank the ‘Associazione Amici del Centro Dino Ferrari’ fortheir support.

Conflict of Interest statement. None declared.

FUNDING

This work was supported by the ARISLA Foundation to G.P.C.‘iPSALS grant’ and MIUR (FIRB RBFR08RV86) to S.C.Funding to pay the Open Access publication charges forthis article was provided by ARISLA (Fondazione Italiana diRicerca per la SLA - Sclerosi Laterale Amiotrofica).

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